1. Field of the Invention
The present invention relates to a polypeptide having ceramidase activity, and a gene encoding said polypeptide. More particularly, the present invention relates to an amino acid sequence of ceramidase as well as a nucleotide sequence encoding the amino acid sequence which are useful as an reagent for lipid engineering for analyzing structure and action of ceramide and as an index for diagnosis of atopic dermatitis. In addition, the present invention relates to a method for producing a polypeptide having ceramidase activity by gene engineering. Further, the present invention relates to a method for detecting the polypeptide, and a method for detecting a gene encoding the polypeptide. Further, the present invention relates to a method for detecting atopic dermatitis by utilizing the above method for detecting the polypeptide and the above method for detecting the gene.
2. Discussion of the Related Art
Ceramidase is an enzyme that hydrolyses ceramide, which is one of sphingolipids, into sphingosine and a fatty acid. Sphingosine formed by hydrolysis of ceramide by ceramidase has various physiological activities, such as inhibition of protein kinase C, activation of phospholipase D, inhibition of a calmodulin-dependent enzyme, and the like, and is an important substance which is considered to have a role in regulating cellular functions through its involvement in cell proliferation and intracellular signal transduction. The regulation of the level of such sphingosine is an important role played by ceramidase.
Ceramidases are classified on the basis of its optimum pH into an acidic ceramidase and a neutral/alkaline ceramidase. There have been reported that ceramidases having optimum pH in an acidic region are present in mammalian tissues such as rat brain [Biochemistry, 8, 1692-1698 (1969)], guinea pig epithelial cell [J. Biol. Chem., 270, 12677-12684 (1995)], human kidney [Biochim. Biophys. Acta, 398, 125-131 (1975)], spleen [Biochim. Biophys. Acta, 1004, 245-251 (1989)], fibroblast [Biochem. J., 205, 419-425 (1982)] and epithelium [FEBS Lett., 268, 110-112 (1990)]; human urine [J. Biol. Chem., 270, 11098-11102 (1995)], and the like.
Among these ceramidases, the amino acid sequences and the nucleotide sequences of ceramidase purified from human urine have been determined [J. Biol. Chem., 271, 33110-33115 (1996)]. Utilizing the homology with this gene, a ceramidase gene of a mouse has also been obtained [Genomics, 50, 267-274 (1998)]. However, all these are acidic ceramidases derived from mammals, and no amino acid sequences or nucleotide sequences of neutral/alkaline ceramidase or of ceramidases derived from microorganisms have yet been determined.
On the other hand, a ceramidase-producing microorganism is known to be present on the epidermis of lesion of atopic dermatitis, and this enzyme is suggested to be causative of or be involved in the exacerbation of atopic dermatitis. However, this enzyme is ceramidase of which optimum pH is within an alkaline region (hereinafter referred to as alkaline ceramidase) [J. Biol. Chem., 273, 14368-14373 (1998)].
When a naturally-occurring ceramidase is produced from a ceramidase-producing microorganism, it is necessary to add an expensive sphingolipid to a culture medium in order to induce the production of an enzyme, and in purification step, it is necessary to separate and remove the remaining sphingolipid or a degradation product thereof from a fraction containing ceramidase. In addition, since enzymes other than ceramidases such as sphingomyelinase are simultaneously produced during the culture, it is difficult to isolate and purify only a desired ceramidase from these enzymes. Moreover, the amino acid sequences or the gene structures of microorganism-derived alkaline ceramidase are completely unknown, so that the method for producing ceramidase by gene engineering cannot be utilized.
Furthermore, although the presence of the ceramidase-producing microorganism is expected to be used as an index for diagnosing atopic dermatitis and as a method for confirming a therapeutic effect, no means for simply detecting or identifying such a microorganism has yet been known. Conventional methods have necessitated extremely complicated processes that the microorganism is isolated and then cultured, and thereafter its ceramidase activity is assayed. Thus, a method for producing a high-purity ceramidase derived from microorganisms at a lower cost is desired as well as a method for detecting a ceramidase-producing microorganism in a simple, non-time-consuming procedure.